PEGylated Molybdenum–Iodine Nanocluster as a Promising Radiodynamic Agent against Prostatic Adenocarcinoma

The combination of photodynamic therapy and radiotherapy has given rise to a modality called radiodynamic therapy (RDT), based on reactive oxygen species-producing radiosensitizers. The production of singlet oxygen, O2(1Δg), by octahedral molybdenum (Mo6) clusters upon X-ray irradiation allows for simplification of the architecture of radiosensitizing systems. In this context, we prepared a radiosensitizing system using copper-free click chemistry between a Mo6 cluster bearing azido ligands and the homo-bifunctional linker bis-dPEG11-DBCO. The resulting compound formed nanoparticles, which featured production of O2(1Δg) and efficient cellular uptake, leading to remarkable photo- and radiotoxic effects against the prostatic adenocarcinoma TRAMP-C2 cell line. Spheroids of TRAMP-C2 cells were also used for evaluation of toxicity and phototoxicity. In vivo experiments on a mouse model demonstrated that subcutaneous injection of the nanoparticles is a safe administration mode at a dose of up to 0.08 g kg–1. The reported results confirm the relevancy of Mo6-based radiosensitizing nanosystems for RDT.


NMR characterizations of the linker
NMR characterizations of Bis-dPEG11-DBCO.For detailed assignment based on gCOSY, HMBC and HSQC experiments see Figures S1A-D.
NMR characterizations of compound 1.Obtained material contained approximately 7% of the unreacted DBCO fragment (estimation based on the integration of 1 H NMR spectra).Peak assignments are based on gCOSY, HMBC and HSQC experiments, analogously with Bis-dPEG11-DBCO experiments.For detailed assignment based on gCOSY, HMBC and HSQC experiments see Figures S1E-H.

In Vivo Toxicity Study
To analyse toxic effects of 1, C57BL/6 (B6), male mice (6 mice per group) were injected subcutaneously with different doses of 1 in the range of 0.1 -2 mg per mouse.One group was only injected with physiological saline solution and left as a control group.Mice were observed daily for signs of toxicity, and their weights were recorded.Eight days after injection, mice were anaesthetized and thereafter autopsied.Blood (25 L) from anesthetised mice was withdrawn using a capillary pipette containing anticoagulant (heparin) from the retroorbital sinus into EDTA-containing tubes.Basic haematological parameters were measured using a Mindray BC-5300 Vet.Abdominal organs (spleen, liver, kidney) were removed for visual examination and imaging by a digital camera.Splenocytes were analysed by flow cytometry for basic immunological cell populations (CD4+, CD8+, CD11b+/Gr-1+, CD69+).Single-cell suspensions were prepared by spleen homogenization through a cell strainer (70 mm; BD Biosciences, San Jose, CA, USA).Erythrocytes were osmotically lysed using ammonium chloride-potassium lysis buffer, and the cell suspension was washed three times in the RPMI-1640 medium and utilised.Selected organs (skin -site of injection, kidney, gallbladder, spleen, lung, liver) were subsequently frozen in liquid nitrogen to analyse Mo concentration in tissue using ICP-MS.

Inductively coupled plasma mass spectrometry
The samples (whole organs or aliquot of homogenized organs) for measurement of the total amount of molybdenum were weighed into a Teflon® vessel, and 3 mL of concentrated 65% nitric acid (high-purity Analpure®, Analytika, Praha, Czech Republic) was added.The digestion procedure was performed using a microwave-heated digestion system Speedwave 4 (Berghof, Eningen unter Achalm, Germany).The temperature was ramped in 8 min to 200 °C and maintained at that temperature for 10 min.The digest was quantitatively transferred into a 50 mL volumetric flask, internal standard indium (Astasol® 1000 ± 2 mg L -1 , Analytika, Praha, Czech Republic) was added in an amount corresponding to a final concentration of 20 μg L -1 , and the flask was filled to mark with water (Milli-Q, Millipore, Bedford, MA, USA).
Molybdenum concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer, Concord, ON, Canada).Quantification was carried out via external calibration.The ICP-MS measurement conditions were as follows: RF power 1.1 kW, nebulizer gas flow rate 0.76 L min -1 , auxiliary gas flow rate 1 L min -1 , plasma gas flow rate 11 L min -1 , measured isotope 98 Mo as an analyte and 115 In as an internal standard.

Figure S2 .Figure
Figure S2.Size distributions by intensity and by number and zeta potentials of fresh (red) and 8-days old (green) PBS dispersions of 1, as obtained by dynamic light scattering.

Figure S4 .
Figure S4.Confocal microscopy images of TRAMP-C2 cells after treatment with 1 and being kept in the dark for 4 hours (A) or after illumination (B).Cells were stained with DAPI (nucleus, cyan) and TMRE (mitochondrial network, magenta), Nile Red (lipids, red (membrane lipids) merged yellow (lipid droplets)) or with LysoTracker (lysosomes, yellow).

Figure S5 .
Figure S5.Photographs of mice.The site of injection is exposed to present the yellow precipitate of 1 under the skin.